E8 media

Two independent clones of the LHX6-mEmerald and LHX6-mCherry reporter lines were used in this study. The parental (isogenic) H7 and KOLF2 lines were used as negative control where relevant. The two clones of mCherry and mEmerald lines behaved indistinguishably in terms of respective fluorescence intensity and pattern and were therefore referred to in short as LHM for the mCherry and LHE for the mEmerald lines.
Routine hPSC culture and MGE differentiation followed procedures described previously [29 (link)]. All hPSCs were cultured on Matrigel-coated plastics in E8 media (Thermo Fisher, Inchinnan, UK). The media was changed daily and the cells passaged mechanically with 0.02% EDTA at 80% confluence. For MGE differentiation, cells from two 80% confluent wells of a 6-well plate were plated onto a 12-well plate previously coated with reduced growth factor Matrigel (VWR) in E8 media (day 0) and changed to N2B27 the next day. The cells were induced to neuroectoderm fate by LDN-193189, SB-431542 and XAV-939 from day 1 to 10, followed by SHH and SHH agonist purmorphamine (PM) induction of ventralization from day 11 to 20. To promote terminal differentiation and cell survival, the cultures were treated with BDNF from day 25 until analysis. Cortical differentiation follows the same procedure without XAV, SHH and PM.

The human embryonic stem cell (hESCs) line (H9; passage 30–35) was a kind gift from Dr. James Thomson (University of Wisconsin-Madison, Madison, WI, USA), and the iPSC line (UTY-1; passage 33–40) was a kind gift from Dr. Ying Liu (University of Texas Health Science Center at Houston, Houston, USA). Both cell lines were cultured in a feeder-free condition on Matrigel-coated 6 well plates (Corning, USA) with E8 media (Thermo Fisher Scientific, USA). Stem cells were passaged by mechanical cutting every 4 days, and the ROCK inhibitor Y- 27632 (10 μM) was added during the first 24 h after passaging.

We generated neurons derived from hiPSCs that are reprogrammed from two healthy donors (one generated in house, and one commercial line; Thermo Fisher Scientific) or PD patients carrying mutations (SNCA ×3) who had given signed informed consent for the derivation of hiPSC lines from skin biopsy as part of the European Union IMI-funded program Stem-BANCC^{37 (link),38 (link)}. The isogenic control line of SNCA ×3 was kindly provided from the Kunath laboratory, and was generated using CRISPR/Cas9 editing^{37 (link)}. PINK1 and the isogenic control lines were purchased from the NINDS Human Genetics Resource Center. The experimental protocol to generate the SNCA ×3 line had approval from the London—Hampstead Research Ethics Committee and R&D approval from University College London, Great Ormond Street Institute of Child Health and the Great Ormond Street Hospital Joint Research Office. The hiPSCs were cultured on Geltrex (Thermo Fisher Scientific) in E8 media (Thermo Fisher Scientific) or mTeSR (Stem Cell Technologies), and passed using 0.5 mM ethylenediaminetetraacetic acid (Thermo Fisher Scientific). All lines were mycoplasma tested (all negative) and short tandem repeat profiled (all matched) performed by the Francis Crick Institute. Neuronal differentiation was performed through dual SMAD inhibition using SB431542 (10 μM, Tocris) and dorsomorphin dihydrochloride (1 μM, Tocris).